The formation of metastases of malignant tumors, which initiate from a primary tumor at more or less remote locations of the body, is one of the most serious problems of tumor therapy, since most of the fatal conditions are caused by such metastases. In recent years there has been considerable success in treating primary tumors by surgery, radiation therapy and chemotherapy. In contrast thereto, the treatment of metastases is extremely difficult and only rarely successful. The risk of metastasis formation is particularly high during the treatment of primary tumors, so that there is an urgent need for preventing the formation of metastases, particularly in this phase.
Exposed cell surface carbohydrate-containing macromolecules have been implicated in growth, morphogenesis, differentiation, recognition, intercellular interactions, and adhesion of tumor cells. Certain surface changes associated with transformation and neoplasia may lead to alterations of the above fundamental processes. Therefore, it is important to study the tumor cell surface characteristics in order to understand factors which influence the expression of the malignant phenotype. The surface properties of tumor cells play a major role in tumor growth at the primary site, invasion into surrounding host tissue, dissemination, embolization, and implantation at distant secondary sites to form metastases. Specifically, in an experimental model of metastasis, that of B16 melanoma (Fidler, Nat. New. Biol. 242: 148-149, 1978; Nicolson et al., Cancer Res. 38: 4105-4111, 1978), it has been shown that tumor cell clumps produce more lung metastases after intravenous injection than do single cells (Fidler, Eur. J. cancer, 9: 223-227, 1973). Further in vitro studies using B16 melanoma variants exhibiting different metastatic potential demonstrated a correlation between the tendency of cells to undergo both homotypic and heterotypic aggregation in vitro, and their metastatic potential in vivo (Fidler et al., Cancer Res, 37: 3945-3956, 1977; Gasic et al., Int. J. Cancer 11: 704-716, 1973; Nicolson et al., Nature (Lond.) 255: 230-232, 1975; Raz et al., Nature (Lond.) 284: 363-364. 1980; Winkelhake et al., J. Nat. Cancer Inst. 56: 285-291, 1976). The homotypic aggregation of B16 melanoma cells depended on the presence of fetal bovine serum. One possible explanation for this requirement could be that a serum glycoprotein(s) mediated intercellular adhesion similar to the action of cell--cell adhesion molecules in other vertebrate systems.
Thorough investigations of metastasis formation, i.e., of organ-specific and non-organ-specific metastases, have resulted in the finding that organ cell lectins are responsible for the formation of metastases. Lectins are highly specific sugar-binding molecules which were first found only in plants, but later on in nearly all other living creatures, including vertebrates. The lectins apparently mainly serve to recognize sugar structures on cell surfaces or in soluble glycoconjugates.
It has further been found that organ cell lectins are responsible for the specific organotropic metastasization. In the course of further intensive investigations it has been found that the formation of metastases of malignant tumors can even be prevented by saturating the lectins of these organ cells with the monosaccharides which are specific for the lectins, and/or with the glycoconjugates containing the monosaccharides in the terminal position.
Monoclonal antibodies have been considered as delivery agents for cytotoxic drugs to treat cancers and to inhibit metastasis of existing cancers. However, there is such a high density of receptors on the surface of cancer cells, and the monoclonal antibodies are such large compounds, that it is impossible to provide sufficient monoclonal antibodies at the cell surface to effectively destroy the cancer cells. The large monoclonal antibodies, in other words, are so large that only a very few can be present at the surface of a cell at any one time.
It is increasingly believed that there is a genetic predisposition to some types of cancer, including some types of colon cancer and some types of breast cancer. Additionally, there are some types of cancers which may be aggravated or caused by a person's behavior or diet, such as smoking causing lung cancer, or a high fat, low fiber diet contributing to the onset of colon cancer. It would be particularly useful for those persons who are at high risk for cancer to be able to act to prevent development of cancer.
Pulverer et al., in U.S. Pat. No. 4,946,830, disclose the use of .beta.-D-galactose and/or glycoconjugates containing terminal .beta.-D-galactose, for inhibiting metastasis of malignant tumors. Other monosaccharides are mannose and glycoconjugates containing a terminally or centrally located mannose as well as L-fucose, N-acetylglucosamine, N-acetylgalactosamine, N-acylneuraminic acids, and derivatives containing neuraminic acid. While the saccharide may be bonded to a carrier, the carrier molecule itself should not be cytotoxically active against tumor cells.
Raz et al., Cancer Research 41: 3642-3647, 1981, disclose that tumor cells include a carbohydrate-binding component(s), which binding was most strongly inhibited by lactose.
Raz et al., ibid., disclose that endogenous lectins on a number of tumor cells exhibited a potent capacity to agglutinate trypsin-treated glutaraldehyde fixed rabbit erythrocytes. This activity was inhibited by millimolar concentrations of lactose, whereas D-galactose, D-galactosamine, and N-acetyl-D-galactosamine were much less potent inhibitors. D-mannose, L-fucose and N-acetyl-O-glucosamine failed to inhibit hemagglutination even at 0.2M.
There have been many reports in the literature relating to the general concept of providing direct transport of an agent which is toxic to tumor cells directly to tumors having .beta.-glucuronidase activity by conjugating the agent with glucuronic acid. Among such reports are Von Ardenne, M. et al., Agressologie, 1976, 176(5): 261-264; East German Patent No. 122,386; German Offenlegungsschrift 22 12 014; Sweeney et al., Cancer Research 31: 477-478, 1971; Baba et al., Gann, 69: 283-284; and Ball, Biochem. Pharm. 23: 3171-3177 (1974).
Von Ardenne et al. suggest many types of aglycones which may be conjugated to glucuronic acid and will be active at the tumor site. These include, broadly, alkylating groups, antimetabolites, cytotoxins, membrane-active (lytic) groups, glycolysis stimulators, respiration inhibitors, inorganic and organic acids and cell cycle stoppers. The East German patent cited above also suggests many such combinations, including 5-fluorouracil-glucuronide, aniline mustard-glucuronide, and many others. The Offenlegungsschrift also mentions a large number of glucuronides. Sweeney et al. disclose the anti-tumor activity of mycophenolic acid-.beta.-glucuronides. Bab et al. note the anti-tumor activity of 5-fluorouracil-o-.beta.-D-glucuronide, and Ball discloses the anti-tumor activity of p-hydroxyaniline mustard glucuronide.
Kneen in European Patent Application 054,924, discloses phenyl ether compounds which can be used to make tumors more sensitive to radiotherapy.
Rubin, in U.S. Pat. Nos. 4,337,760 and 4,481,195, discloses methods for treating tumors having high .beta.-glucuronidase activity with glucuronides with aglycones toxic to the tumor cells with great safety toward the rest of the body by first administering an alkalinizing agent in an amount sufficient to maintain the pH level of non-tumor tissues at approximately 7.5 during the glucuronide treatment to inactivate .beta.-glucuronidase activity in the rest of the body. Thus, the toxic agent is directed only at the cancer cells, as opposed to all of the healthy cells of the body, since the aglycone is only released at the site of the cancer. Tumors having high glucuronidase activity can be identified by assaying tumor cells obtained in a biopsy for .beta.-glucuronidase activity, or by administering a glucuronide whose aglycone has been labelled with a radioactive isotope. If, upon a full body scan, it is found that the radioisotope has accumulated at any specific areas of the body, this will indicate not only the location of the tumor, but the fact that the tumor has sufficient .beta.-glucuronidase activity to deconjugate the glucuronide.
The rationale for the use of 4-hydroxyanisole in the treatment of melanoma is based upon the premise that the only cells in vertebrates that contain tyrosinase are the melanocytes. 4-Hydroxyanisole inhibits DNA synthesis, but by itself shows little toxicity. However, 4-hydroxyanisole is oxidized by tyrosinase to form highly cytotoxic products, and consequently 4-hydroxyanisole is preferentially toxic to those melanoma cells that contain the enzyme tyrosinase [Riley, Philos. Trans. R. Soc. (Biol.) 311: 679, 1985]. Morgan et al., in Clinical Oncology 7: 227-231, 1981, also note that 4-hydroxyanisole, which is oxidized by tyrosinase, gives rise to cytotoxic oxidation products. The specific melanocytotoxic action of this agent is of particular interest because of its use in treatment of malignant melanoma. It was found that localized malignant melanomas treated by intra-arterial infusion of 4-hydroxyanisole underwent regression, although intravenous administration of the drug was not therapeutically effective. The need to use the intra-arterial route of administration imposes certain limits on the use of 4-hydroxyanisole, since it is not always possible to perfuse the site occupied by a tumor. However, it is believed that, as an adjunct to the conventional treatment of primary melanoma in accessible sites, 4-hydroxyanisole infusion will reduce the dissemination of metastases.
Kanclerz et al., in Br. J. Cancer 54: 693-698, 1986, reported that animal studies on experimental melanomas have seen variable results with respect to the therapeutic efficacy of phenolic depigmentation agents. The most active melanocytotoxic agent was found to be an analog of tyrosine, 4-hydroxyanisole. However, evidence for an antitumor effect of 4-hydroxyanisole on melanoma in vivo was found to be variable and not conclusive.
Unfortunately, intra-arterial infusion of 4-hydroxyanisole has serious clinical drawbacks, including difficulties in placing and maintaining the patency of intra-arterial catheters. Clogging and/or clotting frequently occur, and, further more, 4-hyroxyanisole has a short half-life in blood, only about nine minutes, after intra-arterial injection.
Saari, in U.S. Pat. No. 4,812,590, discloses that certain carbamates of 4-hydroxyanisole are suitable substitutes for 4-hydroxyanisole in the treatment of melanoma. These carbamates can be delivered by, for example, intravenous injection, and provide increased levels of 4-hydroxyanisole at the tumor site. The delivery of 4-hydroxyanisole is more convenient and safer than many other methods of delivering 4-hydroxyanisole, although, because serum tyrosinase levels may be elevated in patients having tumors with high tyrosinase activity, the metabolic products of 4-hydroxyanisole may be present in locations other than the tumor site.
Pavel et al., Pigment Cells Research 2: 241-246, 1989, reported an investigation of the human metabolism of 4-hydroxyanisole using urine samples from melanoma patients treated with 4-hydroxyanisole. The most important metabolite of 4-hydroxyanisole was found to be 3,4-dihydroxyanisole, although other metabolic products included 3-hydroxy-4-methoxyanisole and 4-hydroxy-3-methoxyanisole, as well as quinone. These compounds were excreted predominantly as sulfates and glucuronides. Unfortunately, when tyrosinase oxidizes 4-hydroxyanisole in the body, the product, 4-methoxybenzoquinone, is extremely toxic. Because the 4-hydroxyanisole is not confined to the tumor site, and because the serum level of tyrosinase of patients suffering from tyrosinase-active tumors tends to be elevated, there is always the danger in administering 4-hydroxyanisole to such patients whereby an excess of metabolic products of 4-hydroxyanisole will be present in the blood, and thus exert a cytotoxic effect on cells other than tumor cells.
Chen et al. discovered that serum tyrosinase activity in many persons with metastatic diseases was significantly higher than activity in normal persons. Although the highest serum tyrosinase activity was observed in melanoma and breast carcinoma, there is measurable tyrosinase activity in a variety of other metastatic diseases, including lung carcinoma, colon carcinoma, testicular carcinoma, hepatic carcinoma, pancreatic carcinoma, ovarian carcinoma, leukemia, bronchogenic carcinoma, prostate carcinoma, Hodgkin's disease, and rectal carcinoma, the tyrosinase activity of the foregoing diseases listed in decreasing order.
In addition, serum melanin bands were demonstrated by polyacrylamide disc gel electrophoresis of serum tyrosinase followed by incubation of the gel with L-dopa at room temperature overnight to form melanin bands. The following types of metastatic disease demonstrated serum melanin bands with this technique: mouth carcinoma, multiple myeloma, carcinoma of the stomach, carcinoma of the larynx, carcinoma of the cervix, carcinoma of the tonsil, lymphoma, lymphosarcoma, thyroid carcinoma, carcinoma of cecum, endometrial carcinoma, polycytehmia, thymoma, lymphadenopathy, and vertebral carcinoma.
Although the elevation of serum tyrosinase level is explicable in some diseases such as melanoma and breast carcinoma, the high tyrosinase content in melanoma and breast skin increases the tyrosinase circulation level in the blood. Although it has not yet been determined if malignant disease causes a high yield of serum tyrosinase or if a high yield of serum tyrosinase causes malignant disease, it has been postulated that serum immunoglobulins are involved as tyrosinase carriers. Whatever the involvement of tyrosinase in metastatic diseases, there is an elevated level of serum tyrosinase in the case of a great many metastatic diseases.
Passi et al., in Biochem. J. 245: 536-542, 1987, compressed the cytotoxicity of a number of phenols in vitro. These researchers found that in vitro, two melanotic human melanoma cell lines, IRE1 and IRE2, and the lymphoma- and leukemia-derived cell lines Raji and K652, exhibited no significant differences in percentage survival among the different cell lines for each drug tested. The major component of toxicity up to 24 hours of di- and tri-phenols was due to toxic oxygen species acting outside the cells, and not to cellular uptake of these phenols per se. It is believed that scavenger enzymes may interfere with the cytotoxic effect of some of these phenols. Additionally, it was noted that the cytotoxic effect of these phenols was not necessarily related to their being substrates for tyrosinase, as the level of toxicity of butylated hydroxyanisole, which is not a substrate of tyrosinase, was significantly higher than that of 4-hydroxyanisole, which is a substrate of tyrosinase.
With respect to dosages of 4-hydroxyanisole to be given, Wallerie et al. report in "Non-Specific Inhibition of In Vitro Growth of Human Melanoma Cells, Fibroblasts and Carcinoma Cells by 4-Hydroxyanisole" in Hydroxyanisole: Recent Adv. Anti-Melanoma Ther., pp. 153-164 (1984) Editor, Patrick A. Riley, that 4-hydroxyanisole was inhibitory to cultures of human melanotic and amelanotic melanoma cell lines, human fibroblasts and a human bladder carcinoma at concentrations of 10.sup.-3 M to 10.sup.-5 M. This activity was independent of tyrosinase activity, as high tyrosinase activity was only connected with the melanotic cell line, Unfortunately, the therapeutic concentration of 4-hydroxyanisole is difficult to obtain in tissue by intra-arterial infusion of the drug. Furthermore, infusion is given only for one hour twice a day, which is an exposure of the cells that in vitro has no inhibitory effect, even at a high concentration of 4-hydroxyanisole.
It has also been found that a genetic aberration in chromosomes 7 and 13 of certain malignant growths expresses itself in a vast biosynthesis of two specific enzymes: .beta.-glucuronidase and tyrosinase. Among these malignant growths are breast cancer, lung cancer, colon caner, melanoma and gastric cancer.
Para-methoxy-phenyl glucuronide damages cancer cells by excessive production of hydrogen peroxide. Hydrogen peroxide oxidizes many amino acid side chains, such as methionine, by transferring one of the oxygen atoms from the hydrogen peroxide to an acceptor molecule, resulting in damage to the cells. However, cancer cells as well as other living cells contain reduced glutathione (GSH). Glutathione, a tripeptide made up of glutamic acid, cysteine, and glycine, in its reduced state as GSH, can react with hydrogen peroxide to mitigate the oxidative damage to cell membranes, as shown in the following equation: ##STR1##